Bottom Line:
Seventy-six of these proteins were associated with mRNA splicing, including 22 proteins involved in splice site selection.Specifically, TGF-β1 significantly induced expression of SRp20, and reduced the expression of SRp30C, which has been suggested to be a prerequisite for generation of alternatively spliced fibronectin.The results show that TGF-β1 induces the expression of proteins involved in mRNA splicing and RNA processing in human lung fibroblasts.

Background: Transforming growth factor-β1 (TGF-β1) is a potent regulator of cell growth and differentiation. TGF-β1 has been shown to be a key player in tissue remodeling processes in a number of disease states by inducing expression of extracellular matrix proteins. In this study a quantitative proteomic analysis was undertaken to investigate if TGF-β1 contributes to tissue remodeling by mediating mRNA splicing and production of alternative isoforms of proteins.

Methodology/principal findings: The expression of proteins involved in mRNA splicing from TGF-β1-stimulated lung fibroblasts was compared to non-stimulated cells by employing isotope coded affinity tag (ICATTM) reagent labeling and tandem mass spectrometry. A total of 1733 proteins were identified and quantified with a relative standard deviation of 11% +/- 8 from enriched nuclear fractions. Seventy-six of these proteins were associated with mRNA splicing, including 22 proteins involved in splice site selection. In addition, TGF-β1 was observed to alter the relative expression of splicing proteins that may be important for alternative splicing of fibronectin. Specifically, TGF-β1 significantly induced expression of SRp20, and reduced the expression of SRp30C, which has been suggested to be a prerequisite for generation of alternatively spliced fibronectin. The induction of SRp20 was further confirmed by western blot and immunofluorescence.

Conclusions: The results show that TGF-β1 induces the expression of proteins involved in mRNA splicing and RNA processing in human lung fibroblasts. This may have an impact on the production of alternative isoforms of matrix proteins and can therefore be an important factor in tissue remodeling and disease progression.

Figure 6: Western blot analysis of SR proteins after stimulation by TGF-β1. (A) HFL-1 cells were stimulated with TGF-β1 (10 ng/mL) for 6, 24, and 48 h. Cell homogenates were separated by SDS-PAGE and after blotting the proteins were detected with an antibody that recognizes a phospho-epitope on a wide range of SR proteins. Bands were visualized by a fluorescent secondary antibody and analyzed on Odyssey® FC imaging system. The most prominent bands represent the splicing factors SRp75, SRp55, SRp40, SRp30a-c, and SRp20. #The band migrating around 30-kDa could consists of several splicing factors with the approximate molecular weight of 30 kDa. (B) Bands were quantified by densitometry and were then related to their respective GAPDH loading control. Values show the relative expression between TGF-β1-stimulated cells compared to untreated. (C) The expression of SRp20 at 6, 24, and 48 h was examined by western blot and bands were quantified with densitometry. The antibody is not directed against phospho-epitope of the protein. Presented values are the intensity of each band relative to the intensity of the loading control: GAPDH. Each value represent mean and SEM from four individual experiments. *P < 0.05.

Mentions:
Phosphorylated SR proteins from TGF-β1-stimulated HFL-1 cells were analyzed by western blots using an antibody as previously described [34,35]. This antibody detects a conserved phospho-epitope on SR proteins, which does not coincide with absolute protein levels. We detected a complete array of SR proteins, including SRp75, SRp55, SRp40, SRp20, and an additional band that corresponds to proteins that co-migrate around 30 kDa (ASF/SF2, SC35, or SRp30c) [35] (Figure 6A and Table 1). TGF-β1 induced an increase of all the detected SR proteins. A moderate increase was observed at 6 h but at 24 and 48 h the increase was more pronounced (Figure 6A and B). Moreover, TGF-β1 triggered a 3.2-fold increase of the SRp75 at 24 h (P < 0.05) that was preserved at 48 h. SRp55 was 3.0-fold increased at 24 h (P < 0.05) and then slightly decreased at 48 h. SRp40 was continuously increased over time from a 3.5-fold elevation at 24 h (P < 0.05) to a 4.2-fold increase at 48 h. There was 3.4-fold increase of the band that was detected at 30 kDa at 24 h (P < 0.05) and the same increase was retained at 48 h. SRp20 was elevated 8.1-fold compared to untreated cells at 24 h (P < 0.05) which declined to 6.7-fold at 48 h (Figure 6B). To verify the TGF-β1-induced increase of SRp20 we performed separate western blots using an antibody that recognizes the actual protein and does not discriminate between phosphorylated and non-phosphorylated variants (Figure 6C). With this antibody there was a 1.8-fold increase of SRp20 at 24 h (P < 0.05) and a 2.4-fold increase at 48 h (P < 0.05), which supports the results from Additional file 1: Table S1.

Figure 6: Western blot analysis of SR proteins after stimulation by TGF-β1. (A) HFL-1 cells were stimulated with TGF-β1 (10 ng/mL) for 6, 24, and 48 h. Cell homogenates were separated by SDS-PAGE and after blotting the proteins were detected with an antibody that recognizes a phospho-epitope on a wide range of SR proteins. Bands were visualized by a fluorescent secondary antibody and analyzed on Odyssey® FC imaging system. The most prominent bands represent the splicing factors SRp75, SRp55, SRp40, SRp30a-c, and SRp20. #The band migrating around 30-kDa could consists of several splicing factors with the approximate molecular weight of 30 kDa. (B) Bands were quantified by densitometry and were then related to their respective GAPDH loading control. Values show the relative expression between TGF-β1-stimulated cells compared to untreated. (C) The expression of SRp20 at 6, 24, and 48 h was examined by western blot and bands were quantified with densitometry. The antibody is not directed against phospho-epitope of the protein. Presented values are the intensity of each band relative to the intensity of the loading control: GAPDH. Each value represent mean and SEM from four individual experiments. *P < 0.05.

Mentions:
Phosphorylated SR proteins from TGF-β1-stimulated HFL-1 cells were analyzed by western blots using an antibody as previously described [34,35]. This antibody detects a conserved phospho-epitope on SR proteins, which does not coincide with absolute protein levels. We detected a complete array of SR proteins, including SRp75, SRp55, SRp40, SRp20, and an additional band that corresponds to proteins that co-migrate around 30 kDa (ASF/SF2, SC35, or SRp30c) [35] (Figure 6A and Table 1). TGF-β1 induced an increase of all the detected SR proteins. A moderate increase was observed at 6 h but at 24 and 48 h the increase was more pronounced (Figure 6A and B). Moreover, TGF-β1 triggered a 3.2-fold increase of the SRp75 at 24 h (P < 0.05) that was preserved at 48 h. SRp55 was 3.0-fold increased at 24 h (P < 0.05) and then slightly decreased at 48 h. SRp40 was continuously increased over time from a 3.5-fold elevation at 24 h (P < 0.05) to a 4.2-fold increase at 48 h. There was 3.4-fold increase of the band that was detected at 30 kDa at 24 h (P < 0.05) and the same increase was retained at 48 h. SRp20 was elevated 8.1-fold compared to untreated cells at 24 h (P < 0.05) which declined to 6.7-fold at 48 h (Figure 6B). To verify the TGF-β1-induced increase of SRp20 we performed separate western blots using an antibody that recognizes the actual protein and does not discriminate between phosphorylated and non-phosphorylated variants (Figure 6C). With this antibody there was a 1.8-fold increase of SRp20 at 24 h (P < 0.05) and a 2.4-fold increase at 48 h (P < 0.05), which supports the results from Additional file 1: Table S1.

Bottom Line:
Seventy-six of these proteins were associated with mRNA splicing, including 22 proteins involved in splice site selection.Specifically, TGF-β1 significantly induced expression of SRp20, and reduced the expression of SRp30C, which has been suggested to be a prerequisite for generation of alternatively spliced fibronectin.The results show that TGF-β1 induces the expression of proteins involved in mRNA splicing and RNA processing in human lung fibroblasts.

Background: Transforming growth factor-β1 (TGF-β1) is a potent regulator of cell growth and differentiation. TGF-β1 has been shown to be a key player in tissue remodeling processes in a number of disease states by inducing expression of extracellular matrix proteins. In this study a quantitative proteomic analysis was undertaken to investigate if TGF-β1 contributes to tissue remodeling by mediating mRNA splicing and production of alternative isoforms of proteins.

Methodology/principal findings: The expression of proteins involved in mRNA splicing from TGF-β1-stimulated lung fibroblasts was compared to non-stimulated cells by employing isotope coded affinity tag (ICATTM) reagent labeling and tandem mass spectrometry. A total of 1733 proteins were identified and quantified with a relative standard deviation of 11% +/- 8 from enriched nuclear fractions. Seventy-six of these proteins were associated with mRNA splicing, including 22 proteins involved in splice site selection. In addition, TGF-β1 was observed to alter the relative expression of splicing proteins that may be important for alternative splicing of fibronectin. Specifically, TGF-β1 significantly induced expression of SRp20, and reduced the expression of SRp30C, which has been suggested to be a prerequisite for generation of alternatively spliced fibronectin. The induction of SRp20 was further confirmed by western blot and immunofluorescence.

Conclusions: The results show that TGF-β1 induces the expression of proteins involved in mRNA splicing and RNA processing in human lung fibroblasts. This may have an impact on the production of alternative isoforms of matrix proteins and can therefore be an important factor in tissue remodeling and disease progression.